Dye Separation - Leica Microsystems

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8 Confocal Application Letter What is the difference between Weak and Strong Dye Separation? Weak: Strong: The weak method of Automatic Dye Separation will move the clouds until they just touch the axes. The strong method of Automatic Dye Separation will move the clouds to coincide with the axes.
2. Dye Separation: Choosing the Right Tool Channel Dye Separation • When pure dyes are present in the sample or references are available • For separation of two fl uorophores with strong emission overlap, excited with the same excitation line • For separation of autofl uorescence Spectral Dye Separation • When reference spectra are available • For lambda-series • For separation of autofl uorescence Manual Dye Separation • When no reference spectra are available • When Automatic Dye Separation (see below) have failed 3. Dye Separation in LAS AF You can fi nd the Dye Separation tool under Process ➀ and the tab Tools ➁. ➀ ➁ Select the Channel Dye Separation tool. To determine the distribution coeffi cients of the fl uorophores (i.e. degree of crosstalk) you need to defi ne reference regions within your image or series. You may use multi-labeled samples with pure dye regions (see paragraph A). Reference regions may also be defi ned on separately acquired single-dye control images (see paragraph B). Note that you have to keep detection parameters identical for the reference images. Automatic Dye Separation: Weak & Strong • When no reference spectra are available; precondition: good signal to noise ratio • Weak method: weak background and noise reduction • Strong method: strong background and noise reduction 3.1 Channel Dye Separation: Step by Step In order to get reasonable results with any of the Dye Separation tools, it is important to have images with good signal to noise ratios. Dye Separation Confocal Application Letter 9
Page 1 and 2: CONFOCAL APPLICATION LETTER reSOLUT
Page 3 and 4: Fig. 2a: Emission spectrum of the g
Page 5 and 6: 1.1 Channel Dye Separation 1.2 Spec
Page 7: 1.4 Automatic Dye Separation: Weak
Page 11 and 12: The histogram visualizes the color
Page 13 and 14: 2. Place the crosshair in the viewe
Page 15 and 16: Separation of two fl uorophores Ori
Page 17 and 18: 2. In the fi eld Measurement Area
Page 19 and 20: 4. Fill in the fi elds ➃ accordin
Page 21 and 22: ➀ 1. Select the Manual ➀ method
Page 23 and 24: 3.4 Automatic Dye Separation: Weak

Dye Separation - Leica Microsystems

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